Polyethylene glycol-modified urate oxidase

Abstract

To provide treatment of hyperuricemia based on urate oxidase.SOLUTION: The present disclosure provides a polyethylene glycol-modified urate oxidase. At least 11 of the following amino acid sites in the urate oxidase have a PEG modification: T1, K3, K4, K30, K35, K76, K79, K97, K112, K116, K120, K152, K179, K222, K231, K266, K272, K285, K291, K293.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to polyethylene glycol-modified urate oxidase. More specifically, the present invention relates to polyethylene glycol-modified urate oxidase, pharmaceutical combinations, the pharmaceutical use of polyethylene glycol-modified urate oxidase, and methods for reducing the immunogenicity of urate oxidase.

Background Art

[0002] Gout is a disease caused by disorders of purine metabolism. Its clinical feature is hyperuricemia, and urate deposits under the skin, in joints, and in the kidneys to form gouty nodules. Purines in the human body undergo a series of changes, and the final product formed is uric acid. When the blood uric acid concentration exceeds 70 mg / L, it causes hyperuricemia. 5% - 12% of patients with hyperuricemia may develop gout. In blood and synovial fluid, when the concentration of sodium urate reaches saturation, it may form microcrystals of sodium urate salt, which can cause gouty arthritis. Over time, chronic hyperuricemia can also deposit around joints, in soft tissues, and in certain organs, producing destructive crystalline urate deposits, which may cause diseases such as gouty acute arthritis, gouty nodular chronic arthritis, and joint deformation. Kidney damage is considered the second most common clinical symptom of gout. The progressive nature of chronic hyperuricemia leads to the deposition of urate in the medulla, renal tubules, and renal interstitium, stimulating the local area and causing an inflammatory reaction, which is called chronic urate nephropathy. Patients with severe hyperuricemia (such as those with certain malignant tumors, especially leukemia and lymphoma) may have a large amount of uric acid deposited in the renal collecting ducts, renal pelvis, renal calyces, and ureters in a short time, resulting in lumen obstruction and urinary retention, causing acute renal failure (uric acid nephropathy).

[0003] In the past few decades, along with the improvement of people's quality of life, changes in diet and lifestyle, and the increasing intake of high-protein and high-purine foods, the number of gout patients has been showing an increasing trend year by year. In Europe, the number of gout patients has approximately doubled in the past 20 years. Currently, the incidence of hyperuricemia and gout in our country has increased to about 2-3%. When there are no clinical symptoms of hyperuricemia, diet control is sufficient. When clinical symptoms caused by it appear, drug treatment is necessary. The common treatment methods currently used clinically include analgesics and anti-inflammatory drugs such as colchicine, ibuprofen, and naproxen, which are mainly used to control the acute attack symptoms of gouty arthritis and relieve local joint pain, swelling, and inflammation of joints; uricosuric agents such as probenecid, sulfinpyrazone, and benzbromarone that promote the excretion of uric acid (ineffective when renal function declines), and drugs that inhibit uric acid synthesis such as allopurinol. Allopurinol is the main treatment drug for patients suffering from tophaceous gout, renal insufficiency, leukemia, and certain genetic diseases. It inhibits xanthine oxidase, preventing hypoxanthine and xanthine from being converted into uric acid. It gradually oxidizes in the human body to produce oxipurinol, which is easily soluble in water, and is excreted in urine. However, it is difficult to treat patients with chronic gout who have formed tophaceous gout with all conventional treatment methods. In addition, after taking the above drugs for a long time, patients will inevitably develop complications such as leukopenia, cardiac dysfunction, liver dysfunction, gastrointestinal irritation, diabetes due to aplastic anemia, and gout.

[0004] Human hyperuricemia is associated with mutations and inactivation of the uricase gene during human evolution. In the mutation, a premature stop codon (Wu X, Lee C C, Muzny D M, Caskey C T. Proc Natl Acad Sci USA. 1989. 86:9412-9416.) is introduced into the coding sequence of the human uricase gene, so humans cannot synthesize active uricase by themselves. As a result, human purine catabolism ends at uric acid (Wu X, Muzny D M, Lee C C, Caskey C T. J Mol Evol. 1992. 34:78-84.). Active uricase in the liver peroxisomes of non-human primates and other mammals can convert poorly soluble urate (~11 mg / 100 ml water) into more soluble allantoin (~147 mg / 100 ml water) and is excreted more effectively by the kidneys (Wortmann R L, Kelley W N. Kelley’s textbook of rheumatology (6th). 2001:1339-1376). In Europe and the United States, uricase (Uricozyme) prepared from Aspergillus flavus has been used for more than 10 years in the treatment of severe hyperuricemia associated with tumor chemotherapy (Zittoun R, Dauchy F, Teilaud C, Barthelemy M, Bouchard P. Ann Med Interne. 1978. 127:479-482.). The recombinant Aspergillus flavus uricase drug ELITEK, developed by Sanofi in France and manufactured by fermentation of brewing yeast, was approved by the FDA in 2002 and is used for the short-term treatment of severe hyperuricemia caused by tumor chemotherapy (Pui C H, Relling M V, Lascombes F, Harrison P L, Struxiano A et al. Leukemia. 1997. 11 :1813-1816.). At the same time, it has also been proven that the injection of ELITEK can reduce the volume of gout nodules (Potaux L, Aparicio M, Maurel C, Ruedas M E, Mart in C L. Nouv Presse Med. 1975. 4 :1109-1112.).In September 2010, Pegloticase, a PEGylated recombinant porcine-derived uricase manufactured by the US company Savient and approved by the FDA, is used for refractory gout, but since it does not solve the problem of immunogenicity, about 50% of patients have no effect in clinical applications.

[0005] Uricase (EC 1.7.3.3) is widely present in microorganisms (Bacillus fastidiosus, Candida monocytogenes, Aspergillus flavus), plants (soybeans, chickpeas), and animals (pigs, cows, dogs, baboons) (Suzuki K, Sakasegawa S, Misaki H, Sugiyama M. J Biosci Bioeng. 2004. 98: 153-158). In the presence of oxygen, it can catalyze uric acid to oxidize allantoin and release carbon dioxide (Retailleau P, Colloc’h, Denis V, Francoise B. Acta Cryst D. 2004. 60: 453-462.).

[0006] Active uricase is a tetrameric protein composed of the same subunits. The molecular weight of each subunit is about 34 kD and it is composed of 301-304 amino acids. The pH value of the highest enzyme activity of uricase in each solution is 8.0 (Bayol A et al. Biophys Chem. 1995. 54: 229-235.). Among all the known sources of uricase, the highest activity is derived from Aspergillus flavus and reaches 27 IU / mg. Next is Bacillus fastidiosus, and its activity remains at 13 IU / mg (Huang S H, Wu T K. Eur J Biochem. 2004. 271: 517-523.). Also, the uricase derived from leguminous plants has an activity of only 2-6 IU / mg. The uricase derived from mammals reaches a porcine uricase activity of 5 IU / mg after recombinant expression, and the baboon uricase activity reaches only 1 IU / mg (Michael H, Susan J.K. 2006. US7056713B1), and the uricase derived from humans has no activity.

[0007] As an application for humans, the high activity of microbial uricase and the low immunogenicity of mammalian uricase have made these two uricase sources the current hotspots in the research on the development and application of recombinant uricase. However, the homology between uricase derived from Aspergillus flavus and putative human-derived uricase is less than 40% (Lee C C, Wu X, Gibbs R A, Cook R G, Muzny D M, Caskey C T. Science. 1988. 239:1288-1291.), and the human body is prone to producing anti-uricase antibodies, the efficacy of Aspergillus flavus uricase rapidly weakens, and at the same time, it causes severe allergic reactions and cannot be used for long-term treatment.

[0008] Therefore, the treatment of hyperuricemia based on urate oxidase requires further development and improvement.

Summary of the Invention

[0009] This application is made based on the inventor's discovery and recognition of the following facts and problems.

[0010] Active urate oxidase is a homotetrameric protein, one-third of the amino acids are highly hydrophobic amino acids, and the tetrameric proteins easily aggregate to form octamers or larger polymers. Molecules with a molecular weight exceeding 100 kDa can effectively induce an immune response in the body. The molecular weight of the unmodified monomeric urate oxidase protein reaches 140 kDa, and multimeric uricase with a larger molecular weight has higher immunogenicity. The human body is prone to producing anti-uricase antibodies, rapidly weakening its efficacy, and at the same time, causing severe allergic reactions and cannot be used for long-term treatment. Covalent modification of proteins with PEG has been proven to reduce the immunogenicity of proteins, increase the solubility of proteins, and extend the half-life of proteins.

[0011] Duke University and Savient may conduct a chimeric uricase study using pigs and baboons as sources (Michael H, Susan J.K. 2006. US7056713B1). The method of this study is to modify the ε-amino group of the lysine residue of pig-derived uricase with 10KDa methoxy-containing polyethylene glycol (10KDa-mPEG-NPC) under conditions where the enzyme activity does not significantly decrease. The resulting modified product is Pegloticase, which was the first to achieve the goal of treating refractory gout in the human body. The inventors of the present invention found that in the above research results, the problem of immunogenicity caused by drugs could not be completely solved. Clinical subjects discovered that the therapeutic effect of uricase disappeared after multiple injections. The inventors speculated that it might be related to the excessive molecular weight of the Pegloticase protein (when using 10kd of PEG, the molecular weight of Pegloticase becomes 540kDa). At the same time, Pegloticase is not suitable for injection and is suitable for intravenous injection, which reduces the compliance of subjects for long-term use and further severely limits its clinical application. So far, there has been no long-acting uricase drug with lower immunogenicity that can be subcutaneously injected.

[0012] An object of the present invention is to solve at least to some extent one of the technical problems in the related art.

[0013] In a first aspect of the present invention, the present invention provides a polyethylene glycol-modified uricase. According to an embodiment of the present invention, the amino acid sites in the uricase are T 1 , K 3 , K 4 , K 30 , K 35 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 179 , K 222 , K 231 , K 266 , K 272, K 285 , K 291 , K 293 At least 11 of them have PEG modification. It should be noted that the "uricase" described in this application should be understood in a broad sense, which refers to the general name of a mixture of uricases produced in the same batch in actual manufacturing practice. The inventor has found that compared with similar commercially available drugs, at least 11 sites of the above amino acid sites of the polyethylene glycol-modified uricase according to the examples of this application have PEG modification. On the premise of ensuring the maximum enzyme activity, the in vivo stability of uricase can be significantly improved, the immunogenicity can be reduced, and the in vivo efficacy after intramuscular injection can reach the equivalent in vivo efficacy after injection of similar commercially available drugs.

[0014] According to the examples of the present invention, the above uricase may further include at least one of the following additional technical features. According to the examples of the present invention, K 30 , K 35 , K 222 and K 231 At least one, two, three or all of the four lysine sites have PEG modification.

[0015] According to the examples of the present invention, the molecular weight of the polyethylene glycol for PEG modification does not exceed 6KD. The inventor has found that by modifying with polyethylene glycol having a molecular weight of 6KD or less, the long-term effect in vivo of the obtained uricase can be further enhanced, and no serious anti-PEG antibodies are produced due to excessive molecular weight, that is, the immunogenicity is further reduced.

[0016] According to the examples of the present invention, the polyethylene glycol has a monomethoxy group or a hydroxyl group.

[0017] According to the examples of the present invention, the polyethylene glycol has a linear or branched structure.

[0018] According to an embodiment of the present invention, the polyethylene glycol and uricase are bonded via an amide bond.

[0019] According to an embodiment of the present invention, the polyethylene glycol is a modified polyethylene glycol, and the modifying group of the modified polyethylene glycol includes at least one selected from N-hydroxysuccinimide, N-hydroxysuccinimide carbonate, N-hydroxysuccinimide acetate, N-hydroxysuccinimide propionate, N-hydroxysuccinimide butyrate, N-hydroxysuccinimide succinate, and p-nitrobenzene carbonate.

[0020] According to an embodiment of the present invention, the modifying group of the modified polyethylene glycol is N-hydroxysuccinimide propionate.

[0021] According to an embodiment of the present invention, the positioning of the amino acid site is positioned based on the amino acid sequence shown in SEQ ID NO: 1. TYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO: 1).

[0022] According to an embodiment of the present invention, the uricase is a polypeptide having an amino acid sequence shown in SEQ ID NO: 1 to 7, or a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity when compared with SEQ ID NO: 1 to 7, or a polypeptide having substitution, deletion and / or addition of one or more amino acids when compared with SEQ ID NO: 1 to 7. MAHYRNDYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLTSRL(SEQ ID NO:2). MYKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:3). MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTAKRKLASKL(SEQ ID NO:4). MAHYHNDYQKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLNSRREYLHGDNSDIIPTDTIKNTVQVLAKFKGIKSIETFAMNICEHFLSSFNHVIRVQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFLKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWEAVRGIVLKKFAGPYDKGEYSPSVQKTLYDIQVLSLSQLPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLTSRL(SEQ ID NO:5). MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHALAKFKGIKSIEAFAVNICQHFLSSFNHVIRTQVYVEEIPWKRLEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFAAQVYCKWRYHQCRDVDFEATWDTIRDVVLEKFAGPYDKGEYSPSVQKTLYDIQVVSLSQVPEIDDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:6). MADYHNNYKKNDELEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIEAFGVNICEYFLSSFNHVIRAQVYVEEIPWKRLEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQCRDVDFEATWGTIRDLVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:7).

[0023] The amino acid sequence shown in SEQ ID NO:1 is the amino acid sequence of a chimeric uricase (pig - baboon) derived from pig and baboon, the amino acid sequence shown in SEQ ID NO:2 is the amino acid sequence of porcine urate oxidase, the amino acid sequence shown in SEQ ID NO:3 is the amino acid sequence of a chimeric urate oxidase (dog - baboon) derived from dog and baboon, the amino acid sequence shown in SEQ ID NO:4 is the amino acid sequence of canine urate oxidase, the amino acid sequence shown in SEQ ID NO:5 is the amino acid sequence of bovine urate oxidase, the amino acid sequence shown in SEQ ID NO:6 is the amino acid sequence of simian urate oxidase, and the amino acid sequence shown in SEQ ID NO:7 is the amino acid sequence of baboon urate oxidase.

[0024] For the purposes of explanation, the positioning of lysine in this application is based on the amino acid sequence shown in SEQ ID NO:1, for example K 4It refers to the lysine at position 4 based on the amino acid sequence shown in SEQ ID NO:1. Uricase having the amino acid sequences shown in SEQ ID NO:1 to 7, or a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity compared to SEQ ID NO:1 to 7, or a polypeptide having one or more amino acid substitutions, deletions and / or additions compared to SEQ ID NO:1 to 7 is structurally homologous. Those skilled in the art can, by comparing sequences, determine that a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity compared to SEQ ID NO:2 to 7 or SEQ ID NO:1 to 7, or a polypeptide having one or more amino acid substitutions, deletions and / or additions compared to SEQ ID NO:1 to 7 has a corresponding site corresponding to the T 1 , K 3 , K 4 , K 30 , K 35 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 179 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 site is determined, and further, PEG modification occurs at the corresponding sites of the compared polypeptides. The polyethylene glycol-modified uric acid oxidase of the present application has the advantages of low immunogenicity, high in vivo stability and suitability for intramuscular injection. The sequence comparison method is a common method used by those skilled in the art. When comparing the sequence identity of isozymes with different sequence origins, the sequence lengths may be different due to mutations, deletions, etc. between different sequences, but those skilled in the art can determine the identity between different sequences by sequence comparison.

[0025] For example, according to an embodiment of the present invention, the site corresponding to the site where the sequence shown in SEQ ID NO:2 is T 1 , K 3 , K 4 , K 30 , K 35 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 179 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 is M 1 , K 9 , K 10 , K 36 , K 41 , K 82 , K 85 , K 103 , K 118 , K 122 , K 126 , K 158 , K 185 , K 228 , K 237 , K 272 , K 278 , K 297 , K 299 and the corresponding site of the site corresponding to the sequence shown in SEQ ID NO:3 to the sequence shown in SEQ ID NO:1 is M 1 , K 3 , K 29 , K 34 , K 75 , K 78 , K 111 , K 115 , K 119 , K 151 , K 178 , K 221 , K 230 , K 265 , K 271 , K 284 , K 290 , K 292including, the corresponding site where the sequence shown in SEQ ID NO:4 corresponds to the site of the sequence shown in SEQ ID NO:1 is M 1 , K 9 , K 10 , K 36 , K 41 , K 82 , K 85 , K 118 , K 122 , K 126 , K 158 , K 185 , K 228 , K 237 , K 272 , K 278 , K 297 , K 299 including, the corresponding site where the sequence shown in SEQ ID NO:5 corresponds to the site of the sequence shown in SEQ ID NO:1 is, M 1 , K 10 , K 36 , K 41 , K 82 , K 85 , K 118 , K 122 , K 126 , K 158 , K 185 , K 228 , K 237 , K 272 , K 278 , K 297 , K 299 including, the corresponding site where the sequence shown in SEQ ID NO:6 corresponds to the site of the sequence shown in SEQ ID NO:1 is, M 1 , K 9 , K 10 , K 36 , K 41 , K 82 , K 85 , K 118 , K 122 , K 126 , K 158 , K 185 , K 228 , K 237 , K 272 , K 278 , K 291 , K 297 , K 299including, the corresponding sites where the sequence shown in SEQ ID NO:7 corresponds to the sequence shown in SEQ ID NO:1 are M 1 , K 9 , K 10 , K 36 , K 41 , K 82 , K 85 , K 118 , K 122 , K 126 , K 158 , K 185 , K 228 , K 237 , K 272 , K 278 , K 291 , K 297 , K 299 including. The inventors have found through experiments that after PEG modification occurs at at least 11 sites of the corresponding sites of the amino acid sequences shown in SEQ ID NOs: 2 to 7 above, the obtained PEG-modified urate oxidase has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection.

[0026] In a second aspect of the present invention, the present invention provides a polyethylene glycol-modified urate oxidase. According to an embodiment of the present invention, the peptide map of the polyethylene glycol-modified urate oxidase, compared with the peptide map of the urate oxidase not modified with polyethylene glycol, the relative ratio of the peak area reduction of at least 11 predetermined peptide segments is 75% or more, preferably 80% or more, more preferably 90% or more. The polyethylene glycol-modified urate oxidase according to an embodiment of the present invention has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection.

[0027] According to an embodiment of the present invention, the above polyethylene glycol-modified urate oxidase can further include at least one of the following additional technical features, According to an embodiment of the present invention, the peptide map of the polyethylene glycol-modified urate oxidase has peak area reduction peptide segments shown in Table 5.

[0028] According to an embodiment of the present invention, the peptide map of the polyethylene glycol-modified uricase is shown in FIG. 6 or FIG. 7.

[0029] In a third aspect of the present invention, the present invention provides a pharmaceutical combination. According to an embodiment of the present invention, the pharmaceutical composition includes the aforementioned uricase. The pharmaceutical composition according to an embodiment of the present invention has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection, and can be used for the treatment or prevention of hyperuricemia-related diseases.

[0030] According to an embodiment of the present invention, the pharmaceutical composition further includes at least one of the following additional technical features. According to an embodiment of the present invention, the pharmaceutical composition further includes other drugs for treating or preventing hyperuricemia-related diseases.

[0031] In a fourth aspect of the present invention, the present invention provides the use of the aforementioned uricase or the aforementioned pharmaceutical composition in the preparation of a drug, and the drug is used for treating hyperuricemia-related diseases and reducing the level of uric acid in the biological fluid of a subject in need thereof. The uricase according to an embodiment of the present invention has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection, and has significant advantages in the treatment of hyperuricemia-related diseases.

[0032] According to an embodiment of the present invention, the above use can further include at least one of the following additional technical features. According to an embodiment of the present invention, the hyperuricemia-related diseases include diseases selected from chronic hyperuricemia, gout, kidney disease, hyperuricemic arthritis, kidney stones, tophus, hypertension, diabetes, hypertriglyceridemia, metabolic syndrome, coronary heart disease, atherosclerosis, and hyperuricemia caused by cancer chemotherapy.

[0033] According to an embodiment of the present invention, the biological fluid is urine or blood.

[0034] In a fifth aspect of the present invention, the present invention provides a method for reducing the immunogenicity of urate oxidase. According to an embodiment of the present invention, the method is the amino acid site in the urate oxidase, T 1 , K 3 , K 4 , K 30 , K 35 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 179 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 , including generating PEG modification at at least 11 of them. According to the method of the embodiment of the present invention, the immunogenicity of urate oxidase can be effectively reduced, and furthermore, the in vivo safety of the obtained urate oxidase is higher and has a more sustained effect.

[0035] According to an embodiment of the present invention, the above method can further include at least one of the following additional technical features, According to an embodiment of the present invention, at least one, at least two, at least three or four of the four lysine sites of K 30 , K 35 , K 222 , K 231 in the urate oxidase are subjected to PEG modification.

[0036] According to an embodiment of the present invention, the molecular weight of the polyethylene glycol for PEG modification does not exceed 6KD.

[0037] According to an embodiment of the present invention, the polyethylene glycol is a modified polyethylene glycol.

[0038] According to an embodiment of the present invention, the modifying group of the modified polyethylene glycol is N-hydroxysuccinimide.

[0039] According to an embodiment of the present invention, the positioning of the amino acid site is positioned with the amino acid sequence shown in SEQ ID NO: 1.

[0040] According to an embodiment of the present invention, the urate oxidase has the amino acid sequence shown in SEQ ID NOs: 1 to 7 or a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity compared to SEQ ID NOs: 1 to 7, or a polypeptide having one or more amino acid substitutions, deletions and / or additions compared to SEQ ID NOs: 1 to 7.

[0041] It is understood that the technical effects provided by the additional technical features of the aforementioned polyethylene glycol-modified urate oxidase apply to the additional technical features of the method for reducing the immunogenicity of the above urate oxidase according to the embodiments of the present invention, and here, the additional technical features of the method for reducing the immunogenicity of the above urate oxidase according to the embodiments of the present invention will not be repeatedly described.

Brief Description of the Drawings

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Embodiments for Carrying Out the Invention

[0043] Hereinafter, embodiments of the present invention will be described in detail, and examples of the above embodiments are shown in the figures. Hereinafter, the embodiments described with reference to the figures are exemplary and are used for interpreting the present invention and should not be understood as limiting the present invention.

[0044] An object of the present invention is to provide a new polyethylene glycol-modified uricase oxidase.

[0045] Another object of the present invention is to provide a method for effectively reducing the immunogenicity of uricase oxidase. This technology can effectively reduce the immunogenicity of uricase oxidase and improve the in vivo safety and stability of uricase oxidase.

[0046] Another object of the present invention is to provide an application of the polyethylene glycol oxyuricase conjugate obtained above, which can achieve the efficacy of continuously and significantly reducing the blood uric acid level in the body and can be used for the treatment of hyperuricemia and gout.

[0047] In one aspect of the present invention, polyethylene glycol-modified uricase is provided.

[0048] As used herein, the terms "uricase" and "uricase" are used interchangeably and refer to the class of enzymes described in the present invention that catalyze the oxidation of uric acid to produce allantoin and hydrogen peroxide. The terms "uricase analog", "uricase analog", and "uricase derivative" are interchangeable, and based on the activity of uricase specifically catalyzing the conversion of uric acid to allantoin and hydrogen peroxide, structural improvements such as substitution, deletion, or addition of some amino acids can be made to the protein structure sequence of uricase, which can achieve, but are not limited to, the reduction of immunogenicity in this example, the improvement of protein stability, and the promotion of further polyethylene glycol modification.

[0049] The uricase is not particularly limited and may be any origin of uricase and its uricase analogs. Representative examples include, but are not limited to, those derived from mammals, microorganisms, plants, etc.

[0050] In another preferred example, the uricase and its uricase analogs are derived from mammals. Preferably, they are the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and more preferably SEQ ID NO:1.

[0051] The uricases from different species described in the present invention can be obtained by various methods including, but not limited to, natural extraction, chemical synthesis, genetic engineering recombinant expression, etc.

[0052] In another preferred example, urate oxidase is recombinantly expressed in a host cell using recombinant technology with the coding sequence of the urate oxidase protein sequence (SEQ ID NO:1).

[0053] In another preferred example, it is prepared by a method of constructing a recombinant expression strain using Escherichia coli or yeast as a host, and more preferably, recombinant expression is performed using Escherichia coli as a host bacterium.

[0054] As used herein, the polyethylene glycol urate oxidase described in the present invention is obtained by covalently modifying urate oxidase with polyethylene glycol. The polyethylene glycol (PEG) refers to a mixture of an ethylene oxide condensation polymer and water, which is represented by the general formula H(OCH2CH2)nOH. This is a hydrophilic polymer with neutral pH, non-toxicity, and high water solubility, and it exhibits a linear or branched structure. Due to the non-toxicity and good biocompatibility of PEG, currently, the FDA has approved the market launch of multiple types of PEG-modified recombinant protein drugs, proving that PEG can be used to reduce the immunogenicity of proteins, increase the solubility of proteins, and extend the half-life of proteins. To bind PEG to a protein, it is necessary to activate one or more ends of PEG, and the corresponding modifying group can be selected and activated according to the modified target protein such as an amino group, a sulfhydryl group, a carboxyl group, or a hydroxyl group.

[0055] In another preferred example, in the present invention, the sites for PEG modification of urate oxidase and uricase analogs are the ε-amino groups of lysine residues, although a small amount of the α-amino groups of the N-terminal lysine residues are modified. Urate oxidase is covalently bonded and connected to the modifying group of PEG via an amino lipid bond, a secondary amino bond or an amide bond. Preferably, a polyethylene glycol molecule binds to urate oxidase to form an amide bond. The modifying group of the polyethylene glycol includes, but is not limited to, N-hydroxysuccinimide, etc., including N-hydroxysuccinimide (NHS), N-hydroxysuccinimide carbonate (SC), N-hydroxysuccinimide acetate (SCM), N-hydroxysuccinimide propionate (SPA), N-hydroxysuccinimide butyrate (SBA), succinic acid N-hydroxysuccinimide (SS), etc., but is not limited to these. The blocking group of polyethylene glycol includes, but is not limited to, a monomethoxy group, an ethoxy group, glucose or a galactose sugar, and is preferably a monomethoxy group.

[0056] In another preferred example, the polyethylene glycol may be linear or branched.

[0057] In another preferred example, the relative molecular weight of the polyethylene glycol used for polyethylene glycol urate oxidase is 6 KD or less, preferably 1 KD to 5 KD, more preferably 2 KD and 5 KD, and most preferably 5 KD. It should be noted that the "relative molecular weight of polyethylene glycol" described in this application refers to the relative molecular weight of polyethylene glycol without a modifying group, which has a general meaning in the art. After PEG is activated by an activating group, the total relative molecular weight is slightly larger than 5 KD, such as within the range of 5 KD + 10%.

[0058] In another preferred example, the polyethylene glycol-modified urate oxidase has the following characteristics: (1) T, which is an amino acid site in urate oxidase 1 , K 3 , K4 , K 30 , K 35 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 179 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 At least 11 of them have PEG modification. (2) On average, one uricase monomer molecule binds to 11 - 13 polyethylene glycol molecules. (3) K located in the uricase sequence of Seq ID NO:1 30 and / or K 35 , and K 222 and / or K 231 is modified by polyethylene glycol binding. (4) Polyethylene glycol uricase has lower in - vivo immunogenicity.

[0059] In another aspect of the present invention, a method for effectively reducing the immunogenicity of uricase is provided, and this technology can effectively reduce the immunogenicity of uricase and improve the in - vivo stability of uricase.

[0060] As used herein, polyethylene glycol - modified uricase is characterized in that the uricase is not particularly limited and may be any origin of uricase and its uricase analogs, and typical examples include, but are not limited to, those derived from mammals, microorganisms, plants, etc.

[0061] In another preferred example, the urate oxidase and its urate oxidase analogs are derived from mammals. Preferably, they have the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and more preferably SEQ ID NO:1.

[0062] The urate oxidases from different species described in the present invention can be obtained by various methods including, but not limited to, natural extraction, chemical synthesis, recombinant expression by genetic engineering, etc.

[0063] In another preferred example, it is prepared by a method of constructing a recombinant expression strain using Escherichia coli or yeast as a host, and more preferably, recombinant expression is carried out using Escherichia coli as a host bacterium.

[0064] The urate oxidase described in the present invention can be recombinantly expressed in Escherichia coli to obtain a large amount of urate oxidase, and the expressed urate oxidase can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, high-purity urate oxidase can be obtained by methods known to those skilled in the art. Examples of these methods include, but are not limited to, centrifugation, sterilization, salting out, ultrafiltration, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, and combinations of various other techniques.

[0065] The urate oxidase obtained above can be covalently bonded to polyethylene glycol via a linking group using methods known in the art.

[0066] In another preferred example, polyethylene glycol modifies lysine residues on the surface of the spatial structure of uricase in a directional manner. Uricase is covalently bonded to the modifying group (also called the active group) of PEG via an amide bond, and the modifying group (also called the active group) of the polyethylene glycol includes, but is not limited to, N-hydroxysuccinimide (NHS), N-hydroxysuccinimide carbonate (SC), N-hydroxysuccinimide acetate (SCM), N-hydroxysuccinimide propionate (SPA), N-hydroxysuccinimide butyrate (SBA), and succinic acid N-hydroxysuccinimide (SS). The blocking group of polyethylene glycol includes, but is not limited to, a monomethoxy group, an ethoxy group, glucose, or a galactose sugar, and is preferably a monomethoxy group.

[0067] In another preferred example, the polyethylene glycol is linear or straight-chain.

[0068] In another preferred example, the relative molecular weight of polyethylene glycol is 6 KD or less, preferably 1 KD to 5 KD, and most preferably 5 KD.

[0069] In another preferred example, the present invention provides a method for preparing polyethylene glycol-modified uricase, which has one or more features. (1) The modification supply molar ratio of uricase to polyethylene glycol is 1:50 to 1:150 (uricase: polyethylene glycol), and preferably, the supply molar ratio modification is 1:55 to 1:95. (2) The binding reaction system is a carbonate buffer solution, and the modified pH range is 9 to 11.

[0070] The above method for preparing polyethylene glycol-modified uricase uses various purification means to obtain high-purity polyethylene glycol-modified uricase.

[0071] In other preferred examples, methods including, but not limited to, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, tangential flow ultrafiltration, or combinations thereof are used for the purification of the modified sample. More preferably, molecular sieve chromatography and tangential flow ultrafiltration are used.

[0072] In another aspect of the present invention, there is provided the above-mentioned polyethylene glycol-modified uricase and its applications. The conjugate can achieve the efficacy of continuously and significantly reducing blood uric acid levels in the body and can be used for the treatment of hyperuricemia and gout.

[0073] The above-mentioned polyethylene glycol uricase is applied by drugs and their compositions for treating chronic hyperuricemia or gout. The main symptoms of the above-mentioned hyperuricemia and gout include, but are not limited to, uric acid nephropathy and gouty arthritis.

[0074] The administration routes of the above-mentioned polyethylene glycol uricase include, but are not limited to, intravenous injection, subcutaneous injection, intramuscular injection, and intraperitoneal injection, etc. Preferably, intravenous injection and intramuscular injection are used, and more preferably intramuscular injection is used.

[0075] The above-mentioned polyethylene glycol uricase has lower in vivo immunogenicity.

[0076] The fact that the above-mentioned polyethylene glycol uricase has low immunogenicity means that after intramuscular injection of polyethylene glycol uricase into the body of a human or animal, the body does not generate antibodies against polyethylene glycol molecules, or generates antibodies against polyethylene glycol molecules with low titers. It does not generate antibodies against uricase.

[0077] The above-mentioned polyethylene glycol uricase has a longer half-life in the body and the efficacy of reducing blood uric acid levels after intramuscular injection.

[0078] According to an embodiment of the present invention, the polyethylene glycol-modified urate oxidase of the present invention can greatly improve the in vivo stability of urate oxidase and reduce its immunogenicity on the premise of maximizing the enzyme activity, and the in vivo efficacy after intramuscular injection can reach the equivalent in vivo efficacy after injection of similar commercially available drugs. Therefore, the polyethylene glycol-modified urate oxidase of the present invention and the pharmaceutical composition containing the polyethylene glycol-modified urate oxidase can be administered when treating or preventing hyperuricemia-related diseases.

[0079] As used herein, the term "administration" refers to introducing a predetermined amount of a substance into a patient by a suitable method. The polyethylene glycol-modified urate oxidase of the present invention can be administered by any common route as long as it can reach the desired tissue. Various methods of administration include, but are not limited to, the desired peritoneal, intravenous, intramuscular, subcutaneous, dermal, oral, topical, nasal, pulmonary, and rectal, although the present invention is not limited to these recited methods of administration. Additionally, the pharmaceutical composition of the present invention can be administered using a specific device for delivering the active ingredient to the target cells.

[0080] The frequency and dosage of administration of the pharmaceutical composition of the present invention can be determined by a plurality of related factors, which include the type of disease being treated, the route of administration, the age, sex, weight, severity of the patient, and the type of drug as the active ingredient.

[0081] The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly improve a symptom associated with a disease or disorder, i.e., an amount that provides a therapeutic effect for a given disorder and dosing regimen. For example, in the treatment of chronic hyperuricemia or gout, a drug or compound that reduces, prevents, delays, inhibits, or blocks any symptom of the disease or disorder is therapeutically effective. A therapeutically effective amount of a drug or compound need not cure the disease or disorder, but provides treatment for the disease or disorder, such that the onset of the individual's disease or disorder is delayed, blocked, or prevented, the symptoms of the disease or disorder are alleviated, the duration of the disease or disorder is altered, e.g., the disease or disorder becomes less severe, or recovery is accelerated.

[0082] The term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or its side effects due to the disease. "Treatment" as used herein covers the treatment of diseases (primarily referring to hyperuricemia-related diseases) in mammals, particularly humans, and includes (a) preventing the disease in an individual who is susceptible to the disease but has not yet been diagnosed with the disease, (b) suppressing the disease, such as delaying the progression of the disease, or (c) alleviating the disease, such as reducing the symptoms associated with the disease. "Treatment" as used herein includes any drug that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, and includes, but is not limited to, administering the polyethylene glycol-modified urate oxidase described herein to an individual in need thereof.

[0083] According to an embodiment of the present invention, the polyethylene glycol-modified urate oxidase or pharmaceutical composition of the present invention can be used in combination with conventional treatment methods and / or therapies, or can be used separately from conventional treatment methods and / or therapies. When the polyethylene glycol-modified urate oxidase or pharmaceutical composition of the present invention is administered by combination therapy with other drugs, it can be administered to an individual sequentially or simultaneously. Alternatively, the pharmaceutical composition of the present invention can include a combination of the polyethylene glycol-modified urate oxidase of the present invention, a pharmaceutically acceptable carrier or excipient, and other therapeutic or prophylactic drugs known in the art.

[0084] The term "average modification degree" refers to the number of PEGs bound to each uricase monomer.

[0085] In this text, unless otherwise specified, the expression "the amino acid site has PEG modification" means that in the three-dimensional structure of the corresponding polypeptide, the PEG molecule covers the amino acid site, so that at least a part of the groups at the amino acid site is not exposed. Those skilled in the art can determine whether a specific amino acid site is modified by a PEG molecule by conventional technical means. For example, it can be understood that it can be identified by referring to the method listed in the part of "Detection of polyethylene glycol modification site" in Example 3 of this application. In short, the method includes: 1) a step of digesting non-polyethylene glycolylated and polyethylene glycolylated uricase with one or more enzymes, for example, single digestion with Lys-C or Trypsin, or double digestion with Lys-C and Trypsin; 2) separating the digested fragments by high-performance liquid chromatography to create chromatograms of non-polyethylene glycolylated and polyethylene glycolylated uricase, that is, peptide maps; and 3) comparing the differences between the peptide maps of non-polyethylene glycolylated and polyethylene glycolylated uricase, and in combination with a predetermined internal standard peptide segment, determining the relative ratio of the decrease or disappearance of the peptide segment peak where the specific amino acid site of polyethylene glycolylated uricase is located, and further determining whether the specific amino acid site in the peptide segment is modified by PEG. Specifically, in Example 3 of this application, the relative ratio of the decrease or disappearance of the peak area of the peptide segment where the specific amino acid site is located can be calculated by the following formula, P(%)=(A2 - A1) / A2×100%, A1 = A0×t where A0 is the actually measured peak area of the peptide segment where the specific amino acid site of the test target modified protein is located, and t is the average value of the peak area ratio of the internal reference peptide segment between the PHC peptide map and the peptide map of the test target modified protein, P(%) indicates the relative ratio of the decrease or disappearance of the peak area of the peptide segment where a specific amino acid site is located. A2 is the peptide segment peak area of the peptide segment where a specific amino acid site is located in the PHC peptide map, and A1 is the peak area of the peptide segment where a specific amino acid site is located in the test target modified protein peptide map after conversion by internal reference.

[0086] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention can be combined with various technical features specifically described in the following (for example, examples), thereby constituting new or preferred technical solutions, which can be more clearly understood by referring to the following examples. For the limitation of the length of this specification, the above examples are only for the purpose of explanation and are not intended to limit the present invention.

[0087] Hereinafter, the embodiments of the present invention will be described in detail, and examples of the above embodiments are shown in the drawings. The embodiments described below with reference to the drawings are exemplary and are used to interpret the present invention and should not be understood as limiting the present invention.

[0088] Example 1 Preparation of Recombinant Uricase

[0089] 1.1 Construction of the Gene and Expression Plasmid for Uricase Expression Based on the E. coli codon usage bias data, in combination with factors such as codon preference and GC content, the cDNA sequence of the uricase protein (code name: PHC) (SEQ ID NO: 1) was designed, the entire gene was synthesized, and it was named the pUC-57-PHC plasmid. Nde I and BamH I were used as the target gene insertion sites, and the pET-30a plasmid was used as the expression vector (pET-30a-PHC).

[0090] 1.2 Transformation of the Expression Plasmid into Bacterial Host Cells The expression vector pET-30a-PHC was introduced into Escherichia coli BL21(DE3) by the CaCl2 method, clones resistant to Kanamycin were screened, high-expression clones were screened, and the original seed bank strain (E3B) was preserved. These steps were carried out according to common methods in the field of molecular biology.

[0091] 1.3 Preparation of Recombinant Uricase The transformed engineering strain was fermented and expressed in a fermenter. The control conditions were initially 30 °C, the pH was about 7.2, and the culture was continued until OD 600 = 30 or more, IPTG was added to 0.5 mmol / L, and induction was continued for 3 h or more to accumulate uricase. Cells were collected by centrifugation and then stored at -15 °C or lower.

[0092] The frozen bacteria were taken and suspended in 25 mmol / L Tris, 5 mmol / L EDTA buffer with a suspension ratio of 1:10 (W / V). After disrupting the bacterial cells under high pressure, the uricase precipitate was collected by centrifugation. After washing the precipitate once with 50 mmol / L NaHCO3, the concentrated uricase precipitate was suspended in 100 mmol / L Na2HCO3 (pH 9.7 - 10.3) buffer with a suspension ratio of 1:50 (W / V), stirred overnight at room temperature to dissolve, and then the supernatant was collected by centrifugation.

[0093] Uricase was further purified by several chromatography steps. The purity detected by SDS-PAGE was 95% or more, and the purity by Superdex 200 column was 95% or more. There was no aggregate form. The protein concentration was measured using the Lowry method, and the activity of uricase was measured using a spectrophotometer. One unit (U) of enzyme activity was defined as the amount required to convert 1 μmol of uric acid per minute under buffer conditions of an optimal reaction temperature of 37 °C and an optimal pH of 9.0.

[0094] Example 2 Preparation of Polyethylene Glycolated Uricase N-propionyl succinimide PEG with a molecular weight of 5KD (5K-PEG-SPA) was dissolved in a 200 mmol / L PEG solution with 2 mmol / L HCl. After dissolution, at a molar ratio of 1:55 to 1:95 (uricase: 5K-PEG-SPA), the molar ratio of uricase to 5K-PEG-SPA was calculated according to the monomer form, that is, the molar ratio of 1:55 to 1:95 refers to the molar ratio of monomeric uricase to 5K-PEG-SPA. Uricase was added to a carbonate buffer solution with a carbonate concentration of 0.1 - 0.3 mol / L and a pH of 10.0. PEG and uricase underwent a binding reaction, and the binding reaction needed to be stirred and reacted at 5 - 30 °C for at least 60 minutes until the degree of PEG binding no longer changed with time. After the reaction was completed, unmodified PEG and by-products were removed from the reaction by ultrafiltration and / or chromatography. An appropriate molecular sieve chromatography medium was used to separate and remove the modified by-products. Finally, 5K-modified polyethylene glycolated uricase (codenamed PU5) was obtained by sterile filtration.

[0095] Example 3 Characterization Analysis of Polyethylene Glycolated Uricase

[0096] 3.1 Detection of Average Modification Degree and Enzyme Activity The protein concentration is measured by the Lowry method, and the activity of polyethylene glycol uricase oxidase is measured with a spectrophotometer. The maximum ultraviolet absorption wavelength of the uricase substrate uric acid is 293 nm, and the maximum ultraviolet absorption wavelength of the product allantoin is 224 nm. Within a certain concentration range, the absorption value of uric acid at 293 nm is proportional to its concentration, and quantitative measurement of uric acid can be performed by the spectrophotometer method. The specific process is as follows: Using an ultraviolet-visible spectrophotometer, adjust the wavelength to 293 nm, turn on the water bath circulation system of the instrument, and keep the temperature at 37 °C. Using sodium tetraborate buffer as a blank control to correct the zero point, take 2.95 ml of substrate reaction solution (0.1 mol / L sodium tetraborate, 100 μmol / L uric acid, pH 9.5, preheated to 37 °C), put it into a quartz cuvette, then add 50 μl of the test substance, mix quickly, and measure the absorption value at 293 nm. Continuously measure the change in absorbance at 293 nm, and calculate the decomposition concentration of uric acid according to C = A / εL (A is the absorbance value of uric acid at 293 nm at a specific concentration, ε is the molar extinction coefficient of uric acid, L is the optical path of the cuvette, and C is the molar concentration of uric acid), calculate the enzyme activity, and the enzyme activity is defined as the amount of enzyme required to convert 1 μmol of uric acid into allantoin per minute as one activity unit (U) when the optimal reaction temperature is 37 °C and the optimal reaction pH is 9.5.

[0097] The average modification degree of polyethylene glycol uricase oxidase is detected using SEC-HPLC tandem UV / RI (a combination of ultraviolet and refractive index detectors). According to the protein, it has a maximum absorption peak at ultraviolet 280 nm, PEG has no absorption at this wavelength, and at the same time, the absorption values of protein and PEG by the differential refractive index detector are proportional to their various concentrations within a certain range. Therefore, the respective contents of PEG and the protein part in polyethylene glycolated uricase oxidase can be obtained by the external standards of the PEG reference substance and the PHC physical and chemical reference substances, and further, the number of PEG molecules on each uricase oxidase monomer, that is, the average modification degree, can be obtained by the following calculation method.

[0098] Average modification degree of PEG urate oxidase = (relative molecular weight of urate oxidase subunit × amount of PEG in the sample) / (relative molecular weight of PEG × amount of protein in the sample).

[0099] PHC physical and chemical reference substances, PEG reference substances, SEC-HPLC-UV / RI detection diagrams of PU5 modified products are shown in FIGS. 1 to 5.

[0100] Table 1 shows the enzyme activity and average modification degree of polyethylene glycol urate oxidase obtained under different feed ratios in Example 2.

Table 1

[0101] As can be seen from Table 1, the average modification degree of the polyethylene glycol urate oxidase of the present application is 11 or more and is stable, and the enzyme activity has a high enzyme activity retention rate compared with unmodified urate oxidase, and the enzyme activity does not decrease, but instead improves, and the enzyme activity is relatively stable. This is completely different from the commercially available drug Krystexx (pegloticase). According to the content disclosed in the MountainView patent (CN1264575C, FIGS. 2A - 3B) and the general knowledge of those skilled in the art, as the modification degree of 5kD PEG increases, the enzyme activity significantly decreases. However, unexpectedly, the polyethylene glycol uricase obtained in the present application has an average modification degree exceeding 11, and the enzyme activity does not significantly change compared with the case where it is not modified. Therefore, the polyethylene glycol urate oxidase of the present application has a higher average modification degree of polyethylene glycol compared with commercially available drugs, and also achieves an unexpected technical effect regarding the retention of enzyme activity. The inventor presumes that this may be caused by the difference in the PEG modification degree or modification site of polyethylene glycol urate oxidase.

[0102] The inventor modified the proteins of SEQ ID NO: 2-7 to obtain a similar technical effect, that is, when the modification degree of 5kd is greater than 11, a PEGylated urate oxidase is obtained without substantial reduction in enzyme activity.

[0103] 3.2 Detection of polyethylene glycol modification sites In the following steps, the inventor detects the modification sites of the urate oxidase obtained in Example 2.

[0104] The PEG modification sites of polyethylene glycol-modified urate oxidase are determined by digesting non-polyethylene glycolylated and polyethylene glycolylated urate oxidase with one or more enzymes, performing chromatographic detection to obtain a chromatogram, that is, confirming the peptide map. Non-polyethylene glycolylated and polyethylene glycolylated urate oxidase can be digested by single digestion (Lys-C or Trypsin) and / or double digestion (using Lys-C and Trypsin together). The fragments digested by the reverse-phase column are separated, and the modification sites of polyethylene glycol urate oxidase are determined by comparing the disappearance or reduction ratio of the peptide segments by internal reference peptide segment correction.

[0105] The principle of analyzing modification sites in the trypsin and Lys-C double digestion quality peptide map is as follows. Lys-C can specifically digest the C-terminus of lysine (K), and trypsin uses basic amino acids - arginine (R) and lysine (K) as digestion sites to specifically digest the C-terminal peptide bond. By comparing the changes in each corresponding peptide segment before and after digestion with PHC and PU5, and combining the internal standard peptide segments, the relative ratio of the decrease or disappearance of the PEG-modified peptide segment can be analyzed and confirmed. Through the relative ratio of the decrease or disappearance of the peptide segment, it can be determined whether the lysine site on the peptide segment is modified by PEG and the modification ratio. It should be noted that PEG modification is a heterogeneous modification. If the modification ratio of a certain site is high, it can be considered that the site is modified.

[0106] Specifically, it is as follows: (1) Sample treatment: Dissolve uricase and polyethylene glycolated uricase in a digestion buffer (25 mmol / L Tris-HCl, 20% acetonitrile, pH 9.0) at 1 mg / ml respectively and dilute. Take 100 μl each, add 2 μl of Lys-C, and digest at 37 °C for 4 hours. That is, transfer the solution to a pancreatin reaction tube (at a ratio of 1:100), digest at 37 °C for 2 hours, continue the reaction with 4 μl of TCEP reduction solution for 30 minutes, and add 10 μl of 1 mol / L hydrochloric acid solution to stop the reaction. (2) Analysis conditions Instrument: Thermo Ultimate 3000 HPLC and MSQ Plus, Chromatography column: Welch Materials μltimate from Yuexu (R) XB-C 18 (4.6 mm × 250 mm, 5 μm), Analysis conditions: Solution A (aqueous solution containing 0.1% TFA), Solution B (acetonitrile solution containing 0.1% TFA), Gradient: 0 - 70 min, B 3 - 70%, LC detection wavelength: 214 nm. Ion source: ESI, Ion type: Cation, Cone voltage: 50 V, Scanning range: 300 - 2000 Da, Scanning time: 1 S, The split flow after the column is approximately 0.3 ml / min. Inject a sample volume of 100 μl and record the chromatogram. (3) Result processing: Compare the chromatograms (peptide maps) of uricase and polyethylene glycolated uricase, and calculate the relative percentage of the area reduction of different peptide segments. (4) The experimental results are shown in Tables 2 - 5, and Figures 6 - 7.

Table 2

Table 3

[0107] The calculation method of the reduction rate of the peak area of the PU5 peptide segment is as follows: Calculate the peak area of the corresponding PU5 peptide segment at the same concentration of PU5 and PHC according to the following formula: A1 = A0 × t A1 is the peak area of the converted PU5 peptide segment of the two internal reference peptide segments, A0 is the actually measured peak area of the PU5 peptide map peptide segment, and t is the average value of the peak area ratio of the PHC peptide map and the PU5 peptide map in the internal reference peptide segments numbered T30 and T31, that is, 0.588.

Table 4

[0108] The relative percentage of the decrease in the peak area of a certain peptide segment in the PU5 peptide map can be calculated by the following formula using the peptide segment peak area after conversion by internal reference and the PHC peptide map peak area. P(%)=(A2 - A1) / A2×100% In the formula, A2 is the peptide segment peak area of a certain peptide segment in the PHC peptide map, and A1 is the peak area of the PU5 peptide segment of the peptide segment after conversion by internal reference.

[0109]

Table 5

[0110] As can be seen from the analysis of the protein sequence (SEQ ID NO: 1) according to this example, the potential sites where urate oxidase is modified are T 1 , K 3 , K 4 , K 17 , K 21 , K 30 , K 35 , K 48 , K 49 , K 66 , K 74 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 155 , K 158 , K 169 , K 179 , K 190 , K 215 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 and has 31 sites such as

[0111] As can be seen from the analysis of the modification sites of the polyethylene glycol-modified urate oxidase obtained in Example 2, as shown in the analysis of Tables 2, 3, 4, 5 and Figure 6, after digestion with PU5, the sites that disappear in more than 90% of the peptide segments are K 3 、K 4 、K 35 、K 97 、K 112 、K 116 、K 120 、K 152 、K 222 、K 266 、K 285 、 and after digestion with PU5, the sites that disappear within the range of 80% - 90% of the peptide segments are K 76 、K 231 、 and in PU5, all of these sites are modified.

[0112] At the same time, the inventor has discovered that the modification sites of the polyethylene glycol-modified urate oxidase of the present application have more modification sites and are significantly different compared to commercially available drugs. For example, through single digestion, the polyethylene glycol-modified urate oxidase of the present application has a disappearance rate of more than 80% of the peptide segments where the four sites of K 30 、K 35 、K 222 and K 231 are located. By analyzing Krystexx (pegloticase), a similar drug commercially available by the method, the peptide segments where these four sites are located hardly disappear, that is, K 30 、K 35 、K 222 and K 231It was discovered that the modification rates at the four sites were much lower than those of the polyethylene glycol-modified urate oxidase of the present application. Further, the polyethylene glycol-modified urate oxidase of the present application has a significantly reduced immunogenicity compared to commercially available drugs, and the inventors presume that this is related to the number and differences of the modification sites. Since the modification sites and the degree of modification are different, the protection of the in vivo immunogenic sites of the enzyme and the exposure of the active center of the enzyme are different, and the above differences may cause different in vivo biological characteristics of the modified enzymes.

[0113] Hereinafter, the in vivo evaluation of the drug animals of the polyethylene glycol-modified urate oxidase (PU5) of the present application will be described in detail. Pegloticase used in the experiment refers to a commercially available similar drug, and the lot number is 5085B.

[0114] Example 4 Research on the Pharmacokinetics of Polyethylene Glycol Urate Oxidase in Vivo

[0115] 4.1 Evaluation of the Efficacy of Polyethylene Glycol Urate Oxidase in Vivo in Model Rats Potassium oxonate drinking water is used in combination with a high-uric acid diet to induce a chronic hyperuricemia model in rats, and the therapeutic effect of polyethylene glycol urate oxidase (PU5) on chronic hyperuricemia in rats is evaluated.

[0116] Forty model rats are selected and randomly divided into 4 groups, namely the model group, the low-dose administration group of polyethylene glycolated uricase (0.3 mg / kg), the medium-dose administration group of polyethylene glycolated uricase (1.0 mg / kg), and the high-dose administration group of polyethylene glycolated uricase (3.0 mg / kg), with 10 rats in each group. Another 10 normal SD rats are selected as the blank control group. The test is modeled continuously for 5 weeks. After 1 week of modeling, intramuscular administration is started, administered once a week, and continuously administered for 4 weeks. The levels of serum uric acid, serum urea nitrogen, and serum creatinine in rats before administration and 7 days after each administration are detected respectively, and the histological changes of rat kidneys are observed after the test.

[0117] As shown in the results of Figure 8, on the 7th, 14th, 21st, 28th, and 35th days after modeling, compared with the blank control group, the blood uric acid levels in the model control group all increased significantly. The rat serum urea nitrogen, creatinine, and uric acid in the model group 7 days after modeling were 2.73 times, 2.40 times, and 7.83 times that of the rats in the blank group, respectively. From the perspective of renal pathology (as shown in Figure 9), the scores of renal tubular dilation, necrosis, inflammation, and fibrosis in the model control group all increased significantly. At the same time, the quantity of urate crystals also increased significantly. Among the test substances, polyethylene glycolylated uricase at high doses all significantly reduced the serum uric acid level, showing a dose-related relationship. From 14 days to 35 days, the average value of the blood uric acid level in the medium-dose group was maintained at 303.80 - 660.60 μmol / L, and the average value of the blood uric acid level in the high-dose group was maintained at 153.70 - 403.40 μmol / L. Compared with the model group, the range of blood uric acid reduction in the medium-dose group was 34.46 - 67.94%, and the range of blood uric acid reduction in the high-dose group was 65.67 - 83.78%. Compared with the model control group, each administration group of polyethylene glycolylated uricase had a significant improvement effect on renal tubular dilation, kidney necrosis, and inflammation.

[0118] 4.2, Single-dose Administration Evaluation of Polyethylene Glycol Uricase in Rats Thirty-six SD rats were taken, with half being female and half being male, and they were randomly divided into 6 groups (shown in Table 6), namely the intravenous injection group and intramuscular injection group of commercially available Pegloticase, the intravenous injection group of polyethylene glycol uricase, and the low, medium, and high (0.5, 1.0, 2.0 mg / kg) dose intramuscular injection groups of polyethylene glycol uricase. The specific administration plan and dosage are shown in Table 6. PK and PD were detected by jugular vein blood sampling.

[0119]

Table 6

[0120] 4.2.1 Comparison of Pharmacokinetics Before administration to SD rats, the serum drug concentration levels of all individuals were lower than the lower limit of quantification (LLOQ: 312.500 ng / mL). Within the range of 0 - 168 h (0 - 7 days), after single intramuscular injection of polyethylene glycolylated uricase injection (PU5) at 0.5, 1.0, and 2.0 mg / kg, the serum drug concentration showed dose-dependence, and the overall level increased with the increase of the administered dose. After more than 168 h, the blood concentration of the pegloticase intramuscular injection group was lower than the lower limit of quantification, while the intramuscular injection group of PU5 could continue to maintain for more than 240 h.

[0121] After administration, the in vivo C max (C 5min ) ratios of female and male SD rats in each group of the intravenous injection and intramuscular injection groups of 1.0 mg / kg Pegloticase, the intravenous injection group of 1.0 mg / kg polyethylene glycolylated uricase injection, and the intramuscular injection groups of 0.5, 1.0, and 2.0 mg / kg polyethylene glycolylated uricase injection were within the range of 0.75 - 0.99, the AUC last ratios were within the range of 0.54 - 0.94, and the AUC 0-∞ ratios were within the range of 0.58 - 0.97. From this, it can be seen that there is no significant gender difference in the exposure level in the body of SD rats between Pegloticase and polyethylene glycolylated uricase (PU5) injection.

[0122] However, when the same dose (1.0 mg / kg) of the commercial drug Pegloticase was administered to SD rats, the AUC last of the intravenous injection group was 426.48 ± 65.34, the AUC last of the intramuscular injection group was 264.19 ± 78.22, the AUC last of the intravenous injection group of PU5 injection was 565.61 ± 161.60, and the AUC last of the intramuscular injection group was 337.86 ± 227.34. At the same dose and under the condition of the same administration method, the AUC last of PU5 was higher than that of the commercial drug Pegloticase.

[0123] When the commercially available drug Pegloticase at the same dose (1.0 mg / kg) was administered to SD rats, the t 1 / 2 (h) in the intravenous administration group was 49.51 ± 8.12, and the t 1 / 2 (h) in the intramuscular administration group was 55.21 ± 13.50. The t 1 / 2 (h) in the intravenous administration group of the PU5 injection solution was 86.12 ± 33.82, and the t 1 / 2 (h) in the intramuscular administration group was 60.45 ± 21.37. Under the conditions of the same dose and similar administration method, the t 1 / 2 (h) of the PU5 injection solution was longer than that of the commercially available drug Pegloticase.

[0124] The above pharmacokinetic results are shown in Tables 7 to 12, and Figures 10 to 12.

Table 7

[0125]

Table 8

[0126]

Table 9

[0127]

Table 10

[0128]

Table 11

[0129]

Table 12

[0130] 4.2.2. Comparison of in vivo efficacy (uric acid) After single intramuscular injection of polyethylene glycolated uricase injection at 0.5, 1.0, and 2.0 mg / kg, the uric acid concentration was maintained at a low level on the 1st and 3rd days after administration. The uric acid levels in each dose group began to recover on the 7th day after administration. The higher the administered dose, the longer the time for uric acid to be maintained at a low level in the body. Compared with the intravenous injection group of the same dose, the time for the serum uric acid in the PU5 intravenous injection group to be maintained at a low concentration level was longer than that in the pegloticase intravenous injection group in all cases. Compared with the intramuscular injection group of the same dose, the time for the serum uric acid in the PU5 intramuscular injection group to be maintained at a low concentration level was longer than that in the pegloticase intramuscular injection group in all cases. Compared with the same dose group, the time for the serum uric acid in the PU5 intravenous injection or intramuscular injection group to be maintained at a low concentration level was longer than that in the pegloticase intravenous injection group or intramuscular injection group in all cases, that is, the time for PU5 to be maintained at a low concentration level in the body was longer than that in pegloticase in all cases. The results are shown in Figure 13.

[0131] 4.3. Evaluation of multiple administrations of polyethylene glycol uric acid oxidase in rats in vivo In this study, it was divided into 4 groups, namely the intravenous injection group and intramuscular injection group of the commercially available drug Pegloticase, the intravenous injection group and intramuscular injection group of polyethylene glycolated uricase injection (PU5). There were 8 rats in each group, with half males and half females, a total of 32 SD rats. The intravenous injection groups of Pegloticase and polyethylene glycolated uricase injection used intravenous injection, and the intramuscular injection groups of Pegloticase and polyethylene glycolated uricase injection used intramuscular injection. The administered dose was 1.0 mg / kg in all cases. It was administered once a week for 4 consecutive times.

[0132] As can be seen from the result analysis SD rats are intravenously / intramuscularly injected with Pegloticase and polyethylene glycolylated uricase injection at a dose of 1.0 mg / kg multiple times. There are no drug-related abnormal changes in the general condition of the rats.

[0133] 4.3.1 Detection of anti-PEG antibodies After continuous administration to SD rats 4 times, before the first administration, anti-PEG antibodies and anti-PHC antibodies are not detected in all individual animals. After the end of the administration, anti-PHC antibodies are not detected in all animals. Anti-PEG antibodies are detected in each group of the Pegloticase intravenous and intramuscular injection groups and the polyethylene glycolylated uricase injection intravenous and intramuscular injection groups, and the positive result ratios are 3 / 8, 1 / 8, 1 / 8, and 1 / 8 respectively. It is found by PEG immunohistochemical examination that the spleen, liver, and kidney of the Pegloticase intravenous injection group and intramuscular injection group show weak positive expression of PEG. No PEG positive expression is found in the polyethylene glycolylated uricase injection intravenous injection group and intramuscular injection group, and the results are shown in Table 13.

[0134] As can be seen from the above analysis, the antibodies generated by PU5 and Pegloticase are not antibodies against the uricase oxidase part, but mainly antibodies against the PEG part, indicating that both can effectively shield the immunogenic sites of uricase oxidase. The generation of PEG antibodies may cause some side effects in the body. According to the results in Table 13, the immunogenicity of the PU5 of the present application is lower than that of the commercially available product pgeloticase.

[0135] As can be seen from the results of PEG antibodies and PEG immunohistochemistry, both PU5 and Pegloticase are superior in the intramuscular injection group than in the intravenous injection group. The anti-PEG antibodies generated in the intravenous injection group, PU5 is superior to Pegloticase. The anti-PEG antibodies generated in the intramuscular injection group, PU5 is superior to Pegloticase.

[0136]

Table 13

[0137] 4.3.2 Pharmacokinetic Detection After multiple intravenous and intramuscular injections of Pegloticase and polyethylene glycolylated uricase injection into SD rats, there is no significant gender difference in the main pharmacokinetic parameters of the animals in each group. After 4 consecutive administrations, the two drugs accumulate slightly in the rats' bodies.

[0138] Multiple intravenous / intramuscular injections of the commercially available drug Pegloticase at the same dose (1.0 mg / kg) were given to SD rats. After the first administration, the absolute bioavailability in the rats' bodies was 51.35% respectively, and after the last administration, the absolute bioavailability in the rats' bodies was 45.98% respectively. Multiple intravenous / intramuscular injections of polyethylene glycolylated uricase injection at the same dose (1.0 mg / kg) were given to SD rats. After the first administration, the absolute bioavailability in the rats' bodies was 58.29% respectively, and after the last administration, the absolute bioavailability in the rats' bodies was 52.60% respectively.

[0139] 4.3.3 Comparison of in vivo Efficacy (Uric Acid) SD rats were continuously injected intravenously and intramuscularly with 1.0 mg / kg of Pegloticase and polyethylene glycolylated uricase injection 4 times (once a week). After each administration, the serum uric acid concentration was maintained at a low level. From 14 days after the last administration, the Pegloticase intramuscular injection group had recovery, and each of the remaining groups had recovery from 18 days after the last administration. Compared with the commercially available drug Pegloticase at the same dose, the maintenance times of the two drug intravenous injection groups were relatively consistent, and the maintenance time of the polyethylene glycolylated uricase injection intramuscular injection group was longer than that of the commercially available drug, that is, the therapeutic effect of PU5 by intramuscular administration is superior to that of Pegloticase.

[0140] The above results are shown in Tables 14 to 17 and Figures 14 to 19.

[0141]

Table 14

[0142]

Table 15

[0143]

Table 16

[0144] In the description of this specification, references such as "one embodiment", "several embodiments", "example", "specific example", or "several examples" mean that the specific features, structures, materials, or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the exemplary recitation of the above terms does not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. Note that, without contradiction, those skilled in the art can combine and combine the different embodiments or examples and the features of different embodiments or examples described in this specification.

[0145] The embodiments of the present invention have been shown and described above. As can be understood, the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for producing polyethylene glycol-modified urate oxidase, comprising subjecting urate oxidase and polyethylene glycol to a coupling reaction, wherein the polyethylene glycol is provided in the form of an acidic solution, and the molar ratio of the urate oxidase to the polyethylene glycol is 1:(50-150), so as to obtain polyethylene glycol-modified urate oxidase. The polyethylene glycol for polyethylene glycol modification is N-propionate succinimide PEG with a molecular weight of 5 KD. After the N-propionate succinimide PEG with a molecular weight of 5 KD is dissolved in hydrochloric acid, it is used for the modification of urate oxidase. As the amino acid sites of the uricase, T １ , K ３ , K ４ , K ３０ , K ３５ , K ７６ , K ７９ , K ９７ , K １１２ , K １１６ , K １２０ , K １５２ , K １７９ , K ２２２ , K ２３１ , K ２６６ , K ２７２ , K ２８５ , K ２９１ , K ２９３ at least 11 of them have PEG modification, The localization of the amino acid site is localized with the amino acid sequence shown in SEQ ID NO:

1. The urate oxidase has the amino acid sequences shown in SEQ ID NO: 1-7, or Has a polypeptide having at least 90% identity as compared with SEQ ID NO: 1-7, or Has a polypeptide having substitution, deletion and / or addition of one or more amino acids as compared with SEQ ID NO: 1-7. A method for producing polyethylene glycol-modified urate oxidase, characterized by the above.

2. The method according to claim 1, characterized in that the molar ratio of the urate oxidase to the polyethylene glycol is 1:(55-95).

3. The method according to claim 1, characterized in that the concentration of the hydrochloric acid is 2 mmol / L.

4. The method according to claim 1, characterized in that the concentration of the polyethylene glycol in the acidic solution is 200 mmol / L.

5. The method according to claim 1, characterized in that the coupling reaction is carried out in a carbonate buffer solution.

6. The method according to claim 5, characterized in that the pH of the carbonate buffer solution is 9-11.

7. The method according to claim 1, characterized in that the coupling reaction is carried out at 5-30°C for at least 60 minutes.

8. The method according to claim 1, characterized in that the urate oxidase has the amino acid sequences shown in SEQ ID NO: 1-4.

9. The polyethylene glycol-modified urate oxidase K ３０ , K ３５ , K ２２２ and K ２３１ has a PEG modification at at least one of four amino acid sites of, The method according to any one of claims 1-8, characterized in that the localization of the amino acid site is localized with the amino acid sequence shown in SEQ ID NO:

1.

10. The method according to any one of claims 1 to 8, characterized in that the relative ratio of the decrease in the peak area having at least 11 predetermined peptide segments in the peptide map of the polyethylene glycol-modified urate oxidase is 75% or more compared to the peptide map of the urate oxidase not modified with polyethylene glycol.

11. The urate oxidase has the amino acid sequence shown in SEQ ID NO: 1, and the peptide map of the polyethylene glycol-modified urate oxidase has the following table compared to the peptide map of the urate oxidase not modified with polyethylene glycol: The method according to claim 9, characterized in that it has the peak area reduction peptide segment shown in and the relative ratio of the corresponding peak area reduction.

12. The uricase has the amino acid sequence shown in SEQ ID NO: 1, and K ３ , K ４ , K ３５ , K ９７ , K １１２ , K １１６ , K １２０ , K １５２ , K ２２２ , K ２６６ , K ２８５ , K ７６ , K ２３１ has PEG modification at the position, and the method according to claim 9 is characterized in that.

13. The peptide map of the polyethylene glycol-modified urate oxidase shows that, compared with the peptide map of the urate oxidase not modified by polyethylene glycol, the peak area reduction of the peptide segment where amino acid positions K ３ 、K ４ 、K ３５ 、K ９７ 、K １１２ 、K １１６ 、K １２０ 、K １５２ 、K ２２２ 、K ２６６ 、K ２８５ are located is 90% or more, and the peak area reduction of the peptide segment where amino acid positions K ７６ 、K ２３１ are located is 80 - 90%. The method according to claim 12, characterized by this.

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